Data processing of RNAseq data

Overview of sample characteristics:

## 191 samples selected for analysis
## 191 files found
## All files match a metadata entry.
## All expected files found.

Sample QC filtering

Samples with a PC1 > 2 (the red lines) and/or PC2 zscore > 2.5 (the green lines) were dropped from analysis. Points in red represent samples that were dropped.

## Warning: `arrange_()` is deprecated as of dplyr 0.7.0.
## Please use `arrange()` instead.
## See vignette('programming') for more help
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_warnings()` to see where this warning was generated.
Removing the following samples:
sample_name pt_ref subject_ref study subject_id process_ref visit_ref sample_type visit_date sample_alias age ethnicity sex disease_class reason_group match_group random_seed plate_id rin concentration cell_type ifc
AC027_22 BPID|023300 OCRD|1453 OCRD 1453 OCRD|1453|2017-02-24 OCRD|1453|2017-02-23 B Cell RNA 2017-02-23 S01453-V08702 50 AI Female RA RA-AI BPID|020101 0.0047414 RNA NARCH_1 9.3 2.8 b_cell IFC1-1970031135
AC060_22 BPID|013709 CE|541425 CE 541425 CE|541425|2010-03-29 CE|541425|2010-03-29 B Cell RNA 2010-03-29 none 29 AA Female control CTRL-AA BPID|023080 0.9362812 RNA NARCH_1 8.9 2.8 b_cell IFC1-1970031135
AC063_32 BPID|014195 CE|541936 CE 541936 CE|541936|2010-06-23 CE|541936|2010-06-22 T Cell RNA 2010-06-22 none 48 AA Female control CTRL-AA BPID|020101 0.6564617 RNA NARCH_2 7.9 2.8 t_cell IFC3-1970031192

Overall correlation between samples

Data quality

MA-plot

This MA plot is of shrunken log2 fold changes (using the apeglm package), in which actual fold changes are adjusted to compensate for the high variablity of genes with few counts.

Dots in red indicate a significant expression difference between Control and SLE.

Dispersion estimates

K-means clustering optimization

To determine the optimal k, the gap statistic was calculated for k = (3,20) (k = 2 was ignored because it just returns the two disease clusters). A higher coefficient means better intracluster simliarity and intercluster differences.

Sample Sanity check

To determine if there is a gross mixup of samples, examine the expression of sex-specific transcripts to ensure that only female-subject samples express “XIST” and male-subject samples "DDX3Y:

These appear to be correct.

## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
##  setting  value                       
##  version  R version 4.0.0 (2020-04-24)
##  os       Ubuntu 18.04.4 LTS          
##  system   x86_64, linux-gnu           
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  ctype    en_US.UTF-8                 
##  tz       Etc/UTC                     
##  date     2020-10-08                  
## 
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
##  package              * version    date       lib source                                   
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##  acepack                1.4.1      2016-10-29 [1] RSPM (R 4.0.0)                           
##  annotate               1.66.0     2020-04-27 [1] Bioconductor                             
##  AnnotationDbi          1.50.3     2020-07-25 [1] Bioconductor                             
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##  rsconnect              0.8.16     2019-12-13 [1] RSPM (R 4.0.0)                           
##  RSQLite                2.2.0      2020-01-07 [1] RSPM (R 4.0.0)                           
##  rstan                  2.19.3     2020-02-11 [1] RSPM (R 4.0.0)                           
##  rstanarm               2.19.3     2020-02-11 [1] RSPM (R 4.0.0)                           
##  rstantools             2.1.0      2020-06-01 [1] RSPM (R 4.0.0)                           
##  rstatix              * 0.6.0.999  2020-09-18 [1] Github (milescsmith/rstatix@6f5e0a6)     
##  rstudioapi             0.11       2020-02-07 [1] RSPM (R 4.0.0)                           
##  rsvd                   1.0.3      2020-02-17 [1] RSPM (R 4.0.0)                           
##  rvcheck                0.1.8      2020-03-01 [1] RSPM (R 4.0.0)                           
##  rvest                  0.3.5      2019-11-08 [1] RSPM (R 4.0.0)                           
##  S4Vectors            * 0.26.1     2020-05-16 [1] Bioconductor                             
##  scales               * 1.1.1      2020-05-11 [1] RSPM (R 4.0.0)                           
##  scatterpie             0.1.4      2019-11-08 [1] RSPM (R 4.0.0)                           
##  sessioninfo            1.1.1      2018-11-05 [1] RSPM (R 4.0.0)                           
##  shiny                  1.4.0.2    2020-03-13 [1] RSPM (R 4.0.0)                           
##  shinyjs                1.1        2020-01-13 [1] RSPM (R 4.0.0)                           
##  shinystan              2.5.0      2018-05-01 [1] RSPM (R 4.0.0)                           
##  shinythemes            1.1.2      2018-11-06 [1] RSPM (R 4.0.0)                           
##  snakecase              0.11.0     2019-05-25 [1] RSPM (R 4.0.0)                           
##  SnowballC              0.7.0      2020-04-01 [1] RSPM (R 4.0.0)                           
##  StanHeaders            2.21.0-3   2020-05-28 [1] RSPM (R 4.0.0)                           
##  statmod                1.4.34     2020-02-17 [1] RSPM (R 4.0.0)                           
##  storr                  1.2.1      2018-10-18 [1] RSPM (R 4.0.0)                           
##  stringi                1.4.6      2020-02-17 [1] RSPM (R 4.0.0)                           
##  stringr              * 1.4.0      2019-02-10 [1] RSPM (R 4.0.0)                           
##  SummarizedExperiment * 1.18.2     2020-07-09 [1] Bioconductor                             
##  survival               3.1-12     2020-04-10 [2] CRAN (R 4.0.0)                           
##  sva                  * 3.36.0     2020-04-27 [1] Bioconductor                             
##  systemfonts            0.2.2      2020-05-14 [1] RSPM (R 4.0.0)                           
##  threejs                0.3.3      2020-01-21 [1] RSPM (R 4.0.0)                           
##  tibble               * 3.0.1      2020-04-20 [1] RSPM (R 4.0.0)                           
##  tidygraph              1.2.0      2020-05-12 [1] RSPM (R 4.0.0)                           
##  tidymodels           * 0.1.0      2020-02-16 [1] RSPM (R 4.0.0)                           
##  tidyposterior          0.0.2      2018-11-15 [1] RSPM (R 4.0.0)                           
##  tidypredict            0.4.5      2020-02-10 [1] RSPM (R 4.0.0)                           
##  tidyr                * 1.1.0      2020-05-20 [1] RSPM (R 4.0.0)                           
##  tidyselect           * 1.1.0      2020-05-11 [1] RSPM (R 4.0.0)                           
##  tidytext               0.2.4      2020-04-17 [1] RSPM (R 4.0.0)                           
##  tidyverse            * 1.3.0      2019-11-21 [1] RSPM (R 4.0.0)                           
##  timeDate               3043.102   2018-02-21 [1] RSPM (R 4.0.0)                           
##  tokenizers             0.2.1      2018-03-29 [1] RSPM (R 4.0.0)                           
##  triebeard              0.3.0      2016-08-04 [1] RSPM (R 4.0.0)                           
##  tune                 * 0.1.0      2020-04-02 [1] RSPM (R 4.0.0)                           
##  tweenr                 1.0.1      2018-12-14 [1] RSPM (R 4.0.0)                           
##  tximport             * 1.16.1     2020-06-05 [1] Bioconductor                             
##  txtq                   0.2.0      2019-10-15 [1] RSPM (R 4.0.0)                           
##  urltools               1.7.3      2019-04-14 [1] RSPM (R 4.0.0)                           
##  uuid                   0.1-4      2020-02-26 [1] RSPM (R 4.0.0)                           
##  uwot                 * 0.1.8      2020-03-16 [1] RSPM (R 4.0.0)                           
##  vctrs                  0.3.1      2020-06-05 [1] RSPM (R 4.0.0)                           
##  viridis              * 0.5.1      2018-03-29 [1] RSPM (R 4.0.0)                           
##  viridisLite          * 0.3.0      2018-02-01 [1] RSPM (R 4.0.0)                           
##  webshot                0.5.2      2019-11-22 [1] RSPM (R 4.0.0)                           
##  WGCNA                * 1.69       2020-02-28 [1] RSPM (R 4.0.0)                           
##  withr                  2.2.0      2020-04-20 [1] RSPM (R 4.0.0)                           
##  workflows            * 0.1.1      2020-03-17 [1] RSPM (R 4.0.0)                           
##  xfun                   0.14       2020-05-20 [1] RSPM (R 4.0.0)                           
##  XML                    3.99-0.3   2020-01-20 [1] RSPM (R 4.0.0)                           
##  xml2                   1.3.2      2020-04-23 [1] RSPM (R 4.0.0)                           
##  xtable                 1.8-4      2019-04-21 [1] RSPM (R 4.0.0)                           
##  xts                    0.12-0     2020-01-19 [1] RSPM (R 4.0.0)                           
##  XVector                0.28.0     2020-04-27 [1] Bioconductor                             
##  yaml                   2.2.1      2020-02-01 [1] RSPM (R 4.0.0)                           
##  yardstick            * 0.0.6      2020-03-17 [1] RSPM (R 4.0.0)                           
##  zip                    2.0.4      2019-09-01 [1] RSPM (R 4.0.0)                           
##  zlibbioc               1.34.0     2020-04-27 [1] Bioconductor                             
##  zoo                    1.8-8      2020-05-02 [1] RSPM (R 4.0.0)                           
## 
## [1] /usr/local/lib/R/site-library
## [2] /usr/local/lib/R/library